In general, the present invention involves the use of tree sap to cryogenically preserve avian and mammalian sperm cells, preferably for use in the poultry industry, birds of prey preservation, and preservation of endangered or threatened avian species. The present invention may also be used in the cattle industry, pig industry, equine industry, and in mammalian veterinary medicine.
Avian spermatozoa have a shape that makes the spermatozoa hard to freeze. The spermatozoa are long and thin and are shaped like a whip. This makes the cells very subject to cryogenic injury because they have a large surface area that can be damaged easily upon freezing or processing. Mammalian sperm will also benefit from the present invention because even though these cells are easier to freeze, they are still subject to damage from the cryogenic processes. (Reference; Avian Semen Cryopreservation: What Are the Biological Challenges? J. A. Long, 2006 Biotechnology and Germplasm Laboratory, Animal and Natural Resources Institute, Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Beltsville, Md. 20705, 2006 Poultry Science Association, Inc. Accepted Sep. 10, 2005)
Currently, avian spermatozoa are frozen using several techniques. One technique uses the addition of a cryoprotectant to a fluid media that suspends and supports the cells. The first step in the procedure is to collect the semen and then add a liquid extender. A semen extender is a liquid diluent which is added to semen to preserve its fertilizing ability. The extender allows the semen to be freighted to the female, rather than requiring the male and female to be near to each other. Special freezing extender also allows cryogenic preservation of sperm (“frozen semen”), which may be transported for use, or used on-site at a later date.
This extender/cell mixture is then placed in a refrigerator to chill the mixture down to a desired temperature that allows the cells to line up the lipid components in their outer cell membrane prior to freezing. This is a form of “cold acclimation” and helps to allow the cells to survive the cryogenic process. The method also reduces the temperature gradient drop that the cells have to go through before they reach the freezing point and reduces the cell damage when being frozen.
Once the cells are chilled/acclimated, the cryoprotectant is added to the extender/cell mix, the mixture is packaged quickly and either flash frozen by quick immersion in the liquid nitrogen, pelletized and flash frozen and then packaged into cryo-vials, or suspended above the liquid nitrogen in the vapors to freeze more slowly before it is immersed in the liquid nitrogen. Both fast and slow freezing can be done based on species requirements. Different cryoprotectants that are added to the mix commonly include DMSO (Dimethyl sulfoxide), MA (Methyl-Acetamide), and DMA (Dimethyl Acetamide). These chemicals act as intracellular cryoprotectants while the non-cell wall-permeable chemicals act as extracellular cryoprotectants. These are also known to damage the cell wall during cryopreservation and this impairs fertility.
A better and more effective way of preserving avian and mammalian semen is needed in the art.